The proposed research consists of two parts. In Part l, the goal is (1) the removal of the ionic strength restriction of the various conventions (the Bates-Guggenheim, the Machines, etc.) for pH standardization of physiological samples such as blood; and the rationalization of an approach to assignment of pH values using the Pitzer theory for the required single ion activity coefficients of the chloride ion in the standard buffers, at an ionic strength of buffers, at an ionic strength of I=0.16, similar to that found in serum; (2) this will establish a self consistent pH scale and standard reference solutions of reproducible pH, and (3) experimental investigations of dissociation equilibria under the "real" conditions at finite ionic strength (as opposed to zero ionic strength), using the most precise electromotive-force measurement techniques with cells and without a liquid junction. Three systems chosen for study at the early stages of the project in isotonic saline solution are blood phosphate buffer (0.008695m KH2PO4 + 0.03043m Na2HPO4); Zwitterionic amino acid blood buffers (0.06m Na-Tricinate), and (0.05m MOPS + 0.05m Na-Moposate). The long term goals are: (a) to determine the dissociation constants (pK) of physiological buffers; (b) to remove the liquid junction potential errors with the aid of a highly reproducible and predictable flowing liquid junction cell, and (c) to greatly improve the state of pH measurements with a universal pH scale and reference standards. In Part 2, the objective is (1) the development of reference standards and self-consistent scales of ionic activities of the common cations and anions in biological media with application to procedures for the determination of ionic activities with ion-selective electrodes, using the modern theory of Pitzer which yields formulas for the separation of mean activities into their ionic components. Utilizing isotonic saline as a simple matrix, two biological Systems are chosen for study in the initial stages: (I) TES (0.O4m), NaTESate (0.O4m); and (2) HEPES (0.O4m), Na HEPESate (0.04m). Soluble proteins will be added to the buffer-salt systems to determine the existence and magnitude of a protein effect. The long term objectives are: (a) to recommend standardization methods of the four most important ion sensors present in plasma (Na+, K+, Ca+2, Cl-); (b) a quantitative measure of the effect of the electrolytes in saline media on the essential ionic processes which regulate hydrogen ions in body fluids under "real" conditions; and (c) to develop "metal ion buffers" with chelating ligands (for example, CDTA) with calcium amalgam electrode as reference standards for use with ISEs in biological media. The proposal is extraordinarily focused and can be completed in three years.